Centrifugal microfluidics deals with handling liquids in the femtoliter to milliliter range in rotating systems. Such systems are mostly polymer single-use cartridges which are used in or instead of centrifugal rotors, for the purpose of automating laboratory processes. Standard laboratory processes, like pipetting, centrifuging, mixing or aliquoting in a microfluidic cartridge, may be implemented here. For this purpose, the cartridges contain channels for fluid guiding and chambers for collecting liquids. The cartridges are subjected to a predefined sequence of rotational frequencies, the so-called frequency protocol, so that the liquids in the cartridges can be moved by means of the centrifugal force.
Centrifugal microfluidic is mainly applied in laboratory analytics and in mobile diagnostics. Such cartridges may be implemented to be centrifugal-microfluidic discs, which are known under the term “Lab-on-a-disk” and “LabDisk” and “Lab-on-CD”, etc., which are employed in special processing devices. Different formats, like microfluidic centrifugal tubes, which are known under the term “LabTube”, for example, may be employed in rotors of already existing standard laboratory devices.
A fundamental basic operation which is to be performed in centrifugal-microfluidic cartridges, is specifically aliquoting a liquid volume into different sub-volumes, so-called aliquots. The robustness and simplicity of handling the process are of utmost importance for using this basic operation in a possible product. In addition, the basic operation is to be realized monolithically so that no additional components or materials which increase the cartridge costs considerably by material costs or additional setup- and connecting techniques (assembling) are involved.
Different applications, like digital PCR (polymerase chain reaction), single-cell methods, counting bacteria by means of fluorescent phages and manufacturing particles in the micrometer range, involve generating a high number of aliquots. Quantities of several hundred up to more than a million aliquots need to be generated.
It is important for many applications to produce aliquots of small sizes (a few microliters to picoliters or femtoliters). This is of particular importance when a certain amount of aliquots is to be generated in order to perform a desired experiment, but the starting volume is limited, like in digital PCR, for example. Frequently, high costs for reagents, expensive purification of sample materials or small quantities of probe materials are limitations for such applications.
Consequently, there is demand for a basic operation for centrifugal microfluidic systems which allows specifically aliquoting a volume to form many aliquots (several hundred up to over a million) of small volumes (a few microliters to femtoliters). A plurality of techniques for generating droplets on pressure-driven microfluidic and centrifugal microfluidic platforms are known already.
Well-known pressure-driven methods for generating droplets of an aqueous solution in oil use a micro-channel system in order to emulsify the aqueous solution in oil. Thus, the aqueous phase flows through a channel into a chamber filled with oil. It displaces the oil and flows up a step onto a plateau. This plateau is divided into channels by a number of walls. The aqueous phase flows through these channels onto the plateau behind. From there, the phase flows to a downstream chamber and generates an emulsion by droplets breaking off at the edge to the chamber. Such methods are described in [6], [9] to [20] and [22], for example.
[8] describes a method in which a pressure-driven generation and transport of gas bubbles in liquids take place by a varying chamber height. Such methods allow generating bubbles of a gaseous phase in a liquid phase using a micro-channel system. The gaseous phase here flows through a channel to a chamber filled with the aqueous phase. The chamber is beveled such that its flat end is located at the mouth to the channel and has the same height as the channel. Driven by a pressure, the gaseous phase flows to the mouth of the channel where a bubble is pushed into the second phase. Caused by the expanding chamber, bubbles of a defined size break off from the liquid tongue and migrate into the chamber in a flow direction, driven by the chamber height expanding and by capillary forces.
[1] and [2] describe a method for pressure-driven generating and for transporting liquid droplets in liquids by a varying chamber height. This method allows generating droplets of a first liquid phase in a second liquid phase using a micro-channel system. Thus, the first phase flows through a channel into a chamber filled with the second phase. The chamber is beveled such that its flats end is located at the mouth to the channel and has the same height as the channel. Driven by a pump, the first phase flows to the mouth of the channel where a liquid tongue is pushed into the second phase. Caused by the expanding chamber, droplets of a defined size break off from the liquid tongue and migrate into the chamber in the flow direction, driven by the expanding chamber height and by capillary forces.
A comparable method is described in [23]. A pressure-operated system, i.e. not a centrifugal one, which comprises a device for generating droplets, is described.
The core component is an expansion for generation droplets. After a first filling with oil, for example, a second phase, like water, is emulsified at the expansion by capillary forces. The size of the droplets is mainly determined by the geometry of the expansion. In addition, a parallelization by a circular arrangement is described.
[3] and [7] disclose a method for centrifugally generating liquid droplets in air. This method allows generating liquid droplets in air using a micro-channel system and subsequently collecting the droplets in an aqueous solution. Thus, the first liquid phase, driven by a centrifugal force, flows through a channel into a capillary at the end of which there is a micro nozzle suspended freely in air. At the end of the capillary, starting from a certain frequency, droplets break off, which fly through the ambient air over a short distance and then impinge on the surface of a liquid in a collector. There, the droplets harden (partly) by a biochemical reaction and are collected. Thus, the collector is applied such that, at rest, it is perpendicular relative to the ground and is only brought to a horizontal position when applying a centrifugal force.
[5] describes a method for centrifugally generating finished liquid volumes on a rotating disk. This method allows generating finished liquid volumes using a micro-channel and micro-well system. A first liquid is introduced into an inlet chamber of a micro-fluidic system on a rotating disc. Due to a centrifugal force, this liquid moves to a chamber having a large number of small wells which fill up with the first liquid. A second immiscible liquid is used in order to displace the supernatant of the first liquid above the walls of the wells. This interrupts the direct contact of the liquid volumes of the first liquid in the wells among one another.
Apparatuses and methods for generating a mixture of two mutually insoluble phases are described in [4] and [21]. A centrifugal microfluidic disk for generating droplets is provided, wherein droplet generation is based on the coat flow principle. Droplets of an aqueous phase break off from a first channel by pinging off by an oil flow from neighboring channels. After neighboring channels have led to the first channel, the first channel expands and the droplets generated reach the expanded portion of the first channel.